AB0049 A RAPID HEMAGGLUTINATION ASSAY FOR ACPA DETECTION IN PATIENT BLOOD
Authors: I. Kruis, A. Van der Heijden, M. Salden, G. Pruijn
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> Anti-citrullinated protein antibodies (ACPA) are the most specific serological marker of rheumatoid arthritis (RA) and are produced by about 75% of RA patients. The presence of ACPA early in the disease, even before onset of clinical symptoms, facilitates early diagnosis and ACPA is among the most prominent classification criteria for RA.<sup>[1]</sup> Importantly, early diagnosis and immediate start of treatment is strongly correlated with improved outcomes. Several ACPA-detection assays are available for clinical use, which nearly all are based on the same principle: ELISA or related assays with cyclic citrullinated peptides or citrullinated proteins.<sup>[2]</sup> While these methods can be automated in modern diagnostic laboratories, they are ill-suited for low volume laboratories or resource-poor environments. <h3>Objectives</h3> The aim of this study was to develop a rapid and easy to perform assay for ACPA detection. The assay is based on ACPA-dependent agglutination of erythrocytes and can be executed with whole blood. <h3>Methods</h3> An agglutination mediator was developed by protein engineering. Addition of this mediator to (diluted) blood samples results in hemagglutination when ACPA are present. After optimization of the assay with RA serum-spiked blood samples, the applicability was assessed by the analysis of fresh blood samples from 100 RA patients and from 100 psoriatic arthritis (PsA) patients as a control group. Anti-CCP2 and RF levels in these patient samples were determined by standardized ELISAs. <h3>Results</h3> The agglutination mediator that was generated is based on a single-chain antibody fragment that binds to glycophorin A,<sup>[3]</sup> one of the major surface proteins of erythrocytes. It is conjugated to a citrullinated peptide that is efficiently recognized by ACPA. In the presence of erythrocytes and ACPA the mediator induces agglutination of the erythrocytes, which can be detected by the naked eye. The addition of the mediator resulted in detectable agglutination in 64% percent of the RA patient samples. Agglutination correlated well with the results obtained with a commercial anti-CCP2 ELISA for ACPA detection. Efficient agglutination was observed with only 7% of the PsA samples. No correlation with rheumatoid factor levels was observed. <h3>Conclusion</h3> An ACPA-dependent hemagglutination mediator was generated. This agglutination mediator allows the rapid and efficient detection of ACPA by hemagglutination in human blood samples. <h3>References</h3> [1]Aletaha, D. et al. 2010 Rheumatoid arthritis classification criteria. Arthritis Rheum. 2010; 62:2569-81. [2]Pruijn, G.J.M. et al. The use of citrullinated peptides and proteins for the diagnosis of rheumatoid arthritis. Arthritis Res Ther. 2010; 12:203. [3]Bigbee, W.L. et al. Binding specificities of eight monoclonal antibodies to human glycophorin A – studies with M<sup>c</sup>M, and M<sup>k</sup>En(UK) variant human erythrocytes and M and MN<sup>V</sup>-type chimpanzee erythrocytes. J Immunol. 1984: 133:3149-55. <h3>Acknowledgements:</h3> NIL. <h3>Disclosure of Interests</h3> Ilmar Kruis: None declared, Annemarie Van der Heijden: None declared, Martin Salden Employee of: CEO/ R&amp;D director Novio Catalpa BV, Ger Pruijn Grant/research support from: Research grant from Novio Catalpa B.V.